Members of the myc family of nuclear proto-oncogenes (c-, N-, and L-myc) play central roles in the control of normal growth and development and in genetic pathways linked to cellular transformation and apoptotic cell death (Evan and Littlewood, Curr. Biol. 3:44-49 (1993); Morgenbesser and DePinho, Semin. Cancer Biol. 5:21-36 (1994)). Accumulating structural, biochemical, and genetic evidence affords the view that the function of Myc family oncoproteins in these diverse processes relates in part to their roles as sequence-specific transcription factors (for reviews see Kato and Dang, FASEB J. 6:30565-30572 (1992); Torres et al., Curr. Opin. Cell Biol. 4:468-474 (1992). Myc family oncoproteins appear to influence the expression of growth-promoting genes, such as those involved in DNA synthesis (Bello-Fernandez et al., Natl. Acad. Sci. USA 90:7804-7808 (1993)), and cell cycle regulation (Jansen-Durr et al., Proc. Natl. Acad. Sci, USA 90:3685-3689 (1993), in a positive manner. Myc may also play a repressive role in a regulation of some genes through interaction with an initiation factor of the general transcriptional machinery (Roy et al., Nature 365:359-361 (1993)).
Myc family proteins possess a multifunctional amino-terminal domain with transactivation potential (Kato et al., Mol. Cell. Biol. 10:5914-5920 (1990)), a region rich in basic amino acid residues responsible for sequence-specific DNA-binding activity (Blackwell et al., Science 250:1149-1151 (1990)), and a carboxy-terminal .alpha.-helical domain required for dimerization with another basic region-helix-loop-helix-leucine zipper (bHLH-LZ) protein, Max (Blackwood and Eisenman, Science 251:1211-1217 (1991); Prendergast et al., Cell 65:395-407 (1991)). Many of the biochemical and biological activities of Myc appear to be highly dependent upon its association with Max (Blackwood and Eisenman, 1991 supra; Prendergast et al., 1991 supra; Kretzner et al., Nature 359:426-4529 (1992); Amati et al., EMBO J. 12:5083-5087 (1993) and Cell 72:233-245 (1993)). In addition to its key role as an obligate partner in transactivation-competent Myc-Max complexes, Max may also repress Myc-responsive genes through the formation of transactivation-inert complexes that are capable of binding the Myc-Max recognition sequence (Blackwood et al., Genes Dev. 6:71-80 (1992); Kato et al., Genes Dev. 6:81-92 (1992); Kretzner et al., supra 1992; Makela et al., Science 256:373-376 (1992); Mukherjee et al., Genes Dev. 6:1480-1492 (1992); Prendergast et al., Genes Dev. 6:2429-2439 (1992); Ayer et al., Cell 72:211-222 (1993); Zervos et al., Cell 72:223-232 (1993)). These complexes include Max-Max homodimers, whose DNA-binding activity is subject to regulation by case in kinase II phosphorylation (Berberich and Cole, Genes Dev. 6:166-176 (1992), and the recently described hererodimers Mad-Max (Ayer et al., supra 1993) and Mxi-Max (Zervos et al., supra 1991). Together, these functionally interactive and structurally related bHLH-LZ proteins comprise an expanding Myc superfamily.
The rat embryo fibroblast (REF) cooperation assay (Land, et al., Nature 304:596-602 (1983)) has been used in order to understand the role of Myc in cell growth and the functional relationships among members of the Myc superfamily. This highly quantitative biological assay takes advantage of the ability of Myc to cooperate with activated H-RAS (Val-12) to effect the malignant transformation of early passage REFs. The REF cooperation assay has proven effective in the evaluation of candidate modulators of myc oncogenic potential, including the Max-associated proteins MAD and MXI (Lahoz et al., Proc. Natl. Acad. Sci. USA 91:5503-5507 (1994)), dominant negative mutants of Myc (Mukherjee et al., 1992 supra; Sawyers et al., Cell 70:901-910 (1992)), retinoblastoma (Rb), and other cell cycle regulators (Serrano et al., Science 267:249-252 (1995)). The functional impact of such modulators correlates well with their biochemical profiles and postulated mechanisms of action. For instance, Rb, which is thought to interact poorly with Myc in vivo. has a minimal suppressive effect on myc/RAS-induced foci formation, while over expression of MAD or MXI leads to a profound reduction in transformation activity in a highly Myc-specific manner (Lahoz et al., 1994 supra).
In the course of investigating the basis of the anti-oncogenic activity of Mxi, the inventors of the present invention identified two mxi mRNAs that arise though alternative RNA processing and that encode proteins with dramatically different abilities to repress myc-induced transformation. The capacity for strong repressive activity was found to correlate with an amino-terminal extension of 36 residues designated the Mxi "repressive domain", that is present in only one of the two Mxi protein forms. The present invention provides a fusion gene encoding a protein comprising the Mxi repressive domain and the Max bHLH-LZ and carboxy terminus which is an unexpectedly potent inhibitor of the oncogenic effect of Myc family oncoproteins.